TGFβ cytokines were discovered by their capacity to stimulate cell colony formation (Roberts A B, et al, Proc Natl Acad Sci USA 78: 5339-43, 1981); because this process is a classic marker of cellular transformation, this molecule was called Transforming Growth Factor beta. Nowadays TGFβ ligands (TGFβ1, TGFβ2, TGFβ3) are recognized as the prototype of multifunctional growth factors. Almost any type of cell in the body produces them and expresses its receptor complex. These molecules are potent inhibitors of the proliferation of epithelial, endothelial and hematopoietic cells (Ravitz, M J et al, Adv Cancer Res 71: 165-207, 1997) and they are one of the most potent regulators of extracellular matrix production and deposition (Massague, J. The Annu Rev Cell Biol 6: 597-641, 1990) and of tissue repair cascade (Roberts A B., and others, 275-308 Plenum, 1996). They also regulate various mechanisms during embryonic development such as cell differentiation, migration and angiogenesis. (Taya, Y. at al, Development 126: 3869-79, 1999; Lidral, A. et al Am J Hum Genet 63: 557-68, 1998)
In the immune system the TGFβ signaling is a very important regulation node. One of its most important functions is maintaining lymphocyte homeostasis and immune tolerance, through inhibition of the proliferation of naïve T cells induced by self-antigens in lymphopenic environments. (Bevan, M. et al, Nat Immunol. 27, 13 (7): 667-73, 2012). This molecule also suppresses or alters the activation, maturation and differentiation of natural killer cells (Laouar, Y. et al Nature Immunol 6: 600-607, 2005), dendritic cells (Luo, X. et al, Proc. Natl Acad. Sci USA 104: 2821-2826, 2007; Bekeredjian Ding, I. et al, Immunology 128: 439-450, 2009), macrophages (Sica, A. et al, Semin. Cancer Biol 18, 349-355, 2008, Torroella. M et al, Cancer Res 69: 4800-09, 2009), neutrophils (Fridlender, Z G et al, Cancer cell 16: 183-194, 2009) and effector and memory T cells (Gorelik, L. & Flavell, R A Nature Med, 7: 1118-1122, 2001; Flavell, R A Immunity 31:131-44, 2009). The TGFβ plays an essential role in the induction, differentiation and maintenance of natural and induced regulatory T cells (CD4+Foxp3+) and TCD4+IL17+(TH17) effector T cells (Kryczek, I. et al, J. Immunol. 178 : 6730-33, 2007; Moo-Young, T A et al. J. Immunother 32: 12-21, 2009; Fantini, M C et al J. Immunol. 172: 5149-53, 2004; Flavell, R. A. Cell 134: 392-404, 2008).
Mature TGFβ ligands are homodimers of 112 amino acid residues. They are derived from a precursor molecule formed by the latency associated pro-peptide (LAP) located at N-terminal and the active domain towards the C-terminus extreme. Both domains are intracellularly separated by proteolysis and the ligands are secreted as inactive precursors, formed by the prodomain reversibly bound to the active domain, thus regulating access to cellular receptors (Geoffrey D. Young and Joanne E. Murphy-Ullrich. JBC Vol 279, No. 36: 38032-39, 2004). It has been postulated that the pro-peptide associated to latency is also important for secretion of the mature domain (Gray A. and A Mason Science 247:1328-30, 1990).
All three isoforms (TGFβ1, TGFβ2, TGFβ3) interact in the plasma membrane with receptors TβRI, TβRII and TβRIII. The latter, also known as Betaglycan, is not expressed in all cell types, and although it is dispensable for the signaling mediated by TGFβ1 and TGFβ3 ligands, constitutes a reservoir of these ligands when TβRII is saturated. (Wang X F et al, Cell 67: 797-805, 1991; Lebrin F. et al Cardiovasc. Res 65: 599-608, 2005). TβRIII forms complexes with TβRI and TβRII receptors presenting the ligand to them. These receptors bind primarily to TβRII and the TβRII/TGFβ complex recruits and activates cooperatively and with high affinity TβRI receptor, resulting in the formation of a signaling hetero-trimeric complex. TGFβ1 and TGFβ3 can bind to TβRII with high affinity (5-30 pm), while TGF-β2 can only do it in the same way in the presence of TβRIII (De Crescenzo et al, J Biol Chem 279: 26013-18, 2004; De Crescenzo et al, J. Mol. Biol 328: 1173-1183, 2003; Groppe et al, Molecular Cell 29, 157-168, 2008).
So far it has not been reported that ligands of TGFβ1, TGFβ2 and TGFβ3 families have the capacity to bind to other type II receptors, which are part of the same protein family TβRII belongs to (Huang F and Y G Chen Cell Biosc Mar. 15, 2:9, 2012). However they can bind with several type I receptors. ALK5 is described as the reference TβRI receptor of its ligand subfamily. After its recruitment into the TβRII/TGFβ complex the phosphorylation of SMAD2/3 proteins is induced (Huang F and Y G Chen Cell Biosc; Mar. 15, 2:9, 2012). ALK1 is activated in response to the formation of the TGF/TβRII complex in endothelial cells and signals by SMAD1 and SMAD5 (Goumans M J et al, Mol Cell 12 (4): 817-28, 2003). In some epithelial cells the SMAD1/5 signaling is induced by receptor ALK2, ALK3 and ALK6 (Daly A C et al, Mol Cell Biol, 28: 6889-6902, 2008). ALK2 is also associated with processes related to in vivo cardiovascular development (Olivey H E et al, Dev Dyn 235 (1): 50-9, 2006). We should highlight that both TβRII and ALK5 are unique to TGFβ1, TGFβ2 and TGF β3 family of ligands while ALK1/2/6/3 are more promiscuous and also shared by Activins and Bone Morphogenetic Proteins (Sebald W. et al, Biol Chem, 385 (8): 697-710, 2004).
ALK5 mediated signaling is associated with various pathogenic mechanisms in certain diseases. In cancer, for example, its role is complex and is associated with the suppression of immune response and promotion of tumor progression. The suppression of immune response occurs mainly during early stages of tumorigenesis. While its role as tumor progressor occurs in advanced stages of the carcinogenesis, through the induction of metastatic invasive phenotypes and the suppression of anti-tumor immune response (Miyazono K. Nat Rev. Cancer 10: 415-24, 2010; Miyazono K. Cancer Sci 101 (2): 306-12, 2010; Hawinkels L J. et al, Growth Factors 29(4):140-52, 2011). Another illness in which of TGFβ's activity is also deleterious are chronic infections caused by pathogens such as HIV, HBV, HCV, CMV, mycobacteria and Trypanosoma cruzi parasite. The TGFβ exerts a negative influence on the protective immune response, allowing growth and survival of these intracellular pathogens (Reed G S. Microbes and Infection 1: 1313-1325, 1999). In many diseases overproduction of TGFβ contributes to pathological excess of fibrotic tissue, which compromises the normal function of the damaged organ. Some examples of pathological excess of fibrotic tissues are pulmonary fibrosis, sarcoidosis, cardiac fibrosis, cardiomyopathy, liver cirrhosis, systemic sclerosis, glomerular sclerosis and primary biliary cirrhosis, among others (Kopp J B et al, Microbes and Infection, 1: 1349-1365, 1999).
Multiple strategies have been designed to inhibit TGFβ signaling. Most inhibitors have been evaluated in preclinical models, although some of them have begun to be tested in various types of cancer and fibrosis in clinical trials (Flavell R A. Nat Rev Immunol. 10(8): 554-67, 2010; Connolly E C et al, Int J Biol Sci 8(7): 964-78, 2012; Hawinkels L J. et al, Growth Factors 29 (4): 140-52, 2011). In the state of the art you may find the following inhibition strategies:    1—Neutralization of the ligands using soluble forms of the extracellular domains of its receptors (US 2002/0004037 and US 2007/0244042), anti-TGFβ antibodies (US 2002/076858, US 2005/0276802) or oligonucleotides that block transduction of cytokine genes (US 2004/0006036 US2007/0155685).    2—Blockade of signaling using small molecules that bind to the kinase domain of TβRI/ALK5 (US 2006/0057145, US 2005/0136043, US 2006/0234911)    3—Receptor II blockade with antibodies that recognize its extracellular domain (US 2010/0119516, US 2009/7579186).
So far an inhibition strategy using muteins of ligands TGFβ as signaling antagonists has not been reported.